Eradication of malignancies arising in the brain has proven to be a formidable task. As an example, gliomas, the most common primary brain tumor, are almost always fatal despite aggressive surgical resection, radiotherapy and chemotherapy; the overall five year survival rate for glioblastoma (GBM), the most malignant glioma, is less than 5.5% and the median survival is approximately one year.
Because of poor survival of patients with GBM and other brain malignancies, novel therapeutic approaches, most notably viral and gene therapy, have been investigated (for reviews, see Markert et al. (1999) Rev. Med. Virology in press; Cobbs et al. (1999) Persp. Neurolog. Surg. in press; Andreansky et al. (1996) Proc. Natl. Acad. Sci. USA 93, 11313-11318). The efficacy of using neuroattenuated replication-competent herpes simplex viruses (HSV) for treatment of primary brain tumors is known. These viruses typically contain one or more mutations within the viral genome, including thymidine kinase (tk) (Martuza et al. (1991) Science 252, 854-856), ribonucleotide reductase (Mineta et al. (1995) Num. Gene Ther. 1, 938-943; Kramm et al. (1997) Hum. Gene Ther. 8, 2057-2068), UTPase (Pyles et al. (1997) Hum. Gene Ther. 8, 533-544) or γ134.5 (Markert et al. (1993) Neurosurg. 32, 597-603; Chambers et al. (1995) Proc. Natl. Acad. Sci. USA 92, 1411-1415). Moderate increases in long-term survival for engineered HSV-treated versus untreated animals have been reported in both syngeneic and xenogeneic murine tumor models of GBM Markert et al. (2000) Rev. Med. Virology 10, 17-30; Martuza et al. (1991) Science 252, 854-856; Markert et al. (1993) Neurosurg. 32, 597-603; Chambers et al. (1995) Proc. Natl. Acad. Sci. USA 92, 1411-1415; Mineta et al. (1994) Gene Ther. 1 Suppl 1:S78, S78; Andreansky et al. (1997) Cancer Res. 57, 1502-1509; Andreansky et al. (1998) Gene Ther. 5, 121-130; Kaplitt et al. (1994) J. Neuro-Onc. 6, 137-147; Yazaki et al. (1995) Cancer. Res. 55, 4752-4756). In addition, Phase I studies in humans with malignant glioma suggest that a multiply mutated HSV (G207) at doses up to 3×109 pfu is safe for intracranial inoculation (Markert, J M; Medlock, M D; Rabkin S D; Gillespie, G Y; Feigenbaum, F; Hunter, W D; Todo, T; Tornatore, C; and Martuza, R L, unpublished data.
Despite these advantages, it seems likely that multiple modalities of therapy will be necessary to eradicate malignant tumors of the central nervous system (CNS) as well as those originating outside the brain. To increase the efficacy of anti-neoplastic therapy, Applicants studied conditionally replicating γ134.5− mutants as vectors for gene therapy. These vectors retain direct oncolytic effects for tumor cells, and, additionally, express foreign genes designed to augment their anti-tumor effects. Initially, conditionally replicating mutants expressing interleukin 4 (IL-4) and IL-10 were studied (Andreansky et al. (1998) Gene Ther. 5, 121-130). These viruses were evaluated in an orthotopic model of murine glioblastoma utilizing syngeneic GL-261 tumors implanted into immunocompetent C57BL/6 mice. In this model, treatment with IL-4 expressing HSV increased survival over treatment with HSV alone, suggesting that cytokine gene therapy may mediate enhanced tumor-specific killing. IL-4 gene therapy has been shown to enhance anti-glioma effects in several gene therapy models (Okada et al. (1999) Gene Ther. 6, 219-226; Wei et al. (1998) J. Neurovirol. 4, 237-241; Benedetti et al. (1997) Num. Gene Ther. 8, 1345-1353). Such effects are TH-2-mediated and have been attributed to CD4+ lymphocytes and other effector cells such as eosinophils (Tseng et al. (1997) J. Immunother. 20, 334-342). While IL-4 was effective in these animal models, generation of a TH-1 response, including induction of a memory response against tumor cells, need to have a more durable anti-tumor effect.
Therefore, Applicants constructed a virus expressing a cytokine with increased potential for a tumor-specific response. Interleukin-12 (IL-12) is a cytokine with potent anti-tumor properties. It is produced by antigen-presenting cells including B lymphocytes, dendritic cells, and monocytes and acts to enhance the cytolytic activity of natural killer (NK) and cytotoxic T lymphocytes (CTL) and the development of a TH-1-type immune response (Caruso et al. (1996) Proc. Natl. Acad. Sci. USA 93, 11302-11306; Bramson et al. (1996) Num. Gene Ther. 7, 1995-2002; Kishima et al. (1998) Brit. J. of Cancer 78, 446-453; Meko et al. (1996) Surgery 120, 274-283; Nishimura et al. (1996) Ann. NY Acad. Sci. 795, 375-378; Tahara et al. (1995) Num. Gene Ther. 6, 1607-1624; Tahara et al. (1995) Gene Ther. 2, 96-106). IL-12 also possesses anti-angiogenic properties, which may represent a second potential mechanism for its anti-tumor activity (Majewski et al. (1996) J. Invest. Derm. 106, 1114-1118; Kerbel et al. (1995) J. Natl. Cancer Inst. 87, 557-586). IL-12 has been demonstrated to produce anti-glioma immune activity in two different rodent models (Toda et al. (1998) J. Immunol. 160, 4457-4464; Kikuchi et al. (1999) Cancer Let. 135, 47-51). While experimental models utilizing IL-12 for gene therapy have been promising, none have utilized IL-12 expressed from a replication-competent vector (Caruso et al. (1996) Proc. Natl. Acad. Sci. USA 93, 11302-11306; Toda et al. (1998) J. Immunol. 160, 4457-4464; Rakhmilevich et al. (1997) Num. Gene Ther. 8, 1303-1311; Bramson et al. (1996) Num. Gene Ther. 7, 333-342; Tahara et al. (1995) J. Immunol. 154, 6466-6474; Myers et al. (1998) Laryngoscope 108, 261-268). Notably, Phase I human studies utilizing systemic IL-12 therapy have demonstrated toxicity of this cytokine, presumably due to its pleitrophic effects (Marshall et al. (1995) Science 1555).
Applicants have developed a conditionally replication-competent, γ134.5− mutant, which expresses murine IL-12 (M002), for treatment of brain tumors that retains its ability to replicate in murine tumor cells, maintains its primary characteristic of direct tumor cell oncolysis, produces IL-12 at physiologically relevant amounts, allows for direct expression of the cytokine within the tumor cells after inoculation, increases survival of A/J mice, a murine strain more sensitive to HSV infection, implanted with a syngeneic immunocompetent clone of Neuro2A neuroblastoma tumor cells (median=52 days) after treatment with M002 versus treatment with the non-cytokine expressing parent virus, R3659 (median=24 days), and which significantly increases immune-related inflammatory infiltration by CD4+ T cells, macrophages and to a lesser extent, CD8+ cells in M002-treated tumors versus R3659-treated tumors in brain tissue.
Applicants have also developed conditionally replication competent, γ134.5 mutants, which express murine GM-CSF (M004) and bacterial CD (M012) for treatment of brain tumors and other cancers.
There exists a need for an anti-tumor therapy, specifically for the treatment of tumors of the central nervous system such as brain tumors and other tumors originating outside the brain with cytokines, that overcomes the problems and disadvantages of previous therapies. The present invention fulfills this long-standing need in the art.